Phosphate-regulated expression of biologically active recombinant coronavirus glycoproteins and other recombinant proteins in phaeodactylum tricornutum

Abstract

Phosphate-regulated expression of recombinant glycoprotein antigens and other recombinant proteins in diatoms is described herein. More specifically, described herein is the expression and purification of glycosylated, immunogenic, and serologically active receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, as well as SARS-CoV-2 nucleocapsid protein, in the marine pennate diatom Phaeodactylum tricornutum , as well as a functional lateral flow assay-based diagnostic device based on the produced recombinant RBD and nucleocapsid protein. Also described herein is the use of phosphate/iron levels in culture media to regulate expression/secretion of recombinant proteins under control of an HASP1 promoter in P. tricornutum or other suitable host cells. Also described herein is a method for increasing the expression/secretion of a recombinant protein by engineering the recombinant protein to lack a Tobacco Etch Virus (TEV) protease cleavage site.

1 . A recombinant glycoprotein or protein comprising a coronavirus polypeptide antigen having a glycosylation or other post-translational modification pattern produced by, or characteristic of, post-translational modification by  Phaeodactylum tricornutum . 
     
     
         2 . The recombinant glycoprotein or protein of  claim 1 , wherein the coronavirus polypeptide antigen:
 (i) has an N-linked glycosylation pattern and/or phosphorylation pattern produced by, or characteristic of, post-translational modification by  P. tricornutum ;   (ii) is a betacoronavirus polypeptide antigen (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV);   (iii) is from a surface glycoprotein or protein (e.g., spike (S) protein, a nucleocapsid (N) protein, a membrane protein, or an envelope protein);   (iv) is or comprises a fragment of a coronavirus spike protein (e.g., a fragment comprising S1 subunit, S2 subunit, or receptor binding domain); or a fragment of a coronavirus nucleocapsid protein (e.g., an N-terminally truncated nucleocapsid protein, such as an N-terminally truncated nucleocapsid protein lacking contiguous residues 19-110, 19-111, 19-112, 19-113, 19-114, 19-115, 19-116, 19-117, 19-118, 19-119, 19-120, 19-121, 19-122, 19-123, 19-124, 19-125, 19-126, 19-127, 19-128, 19-129, 19-130, 19-131, 19-132, 19-133, 19-134, 19-135, 19-136, 19-137, 19-138, 19-139, 19-140, 19-141, 19-142, 19-143, 19-144, 19-145, 19-146, 19-147, 19-148, 19-149, 19-150, 19-151, 19-152, 19-153, 19-154, 19-155, 19-156, 19-157, 19-158, 19-159, 19-160, 19-161, 19-162, 19-163, 19-164, 19-165, 19-166, 19-167, 19-168, 19-169, 19-170, 19-171, 19-172, 19-173, 19-174, 19-175, 19-176, 19-177, 19-178, 19-179, 19-180, 19-181, 19-182, 19-183, 19-184, 19-185, 19-186, 19-187, 19-188, 19-189, 19-190, 19-191, 19-192, 19-193, 19-194, 19-195, 19-196, 19-197, 19-198, 19-199, 19-200, 19-201, 19-202, 19-203, 19-204, 19-205, 19-206, 19-207, 19-208, 19-209, 19-210, 19-211, 19-212, 19-213, 19-214, 19-215, 19-216, 19-217, 19-218, 19-219, 19-220, 19-221, 19-222, 19-223, 19-224, 19-225, 19-226, 19-227, 19-228, 19-229, 19-230, 19-231, or 19-232 of SEQ ID NO: 23);   (v) is or comprises a fragment of a coronavirus spike protein’s receptor binding domain (RBD), wherein: 
 (a) the recombinant glycoprotein competitively inhibits binding of a native RBD protein produced in mammalian (e.g., human) cells to a human ACE2 receptor; and/or 
 (b) the recombinant glycoprotein cross-reacts with antibodies (e.g., neutralizing antibodies) raised against the spike protein or an RBD-comprising fragment thereof, or 
   (vi) comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1, to SEQ ID NO: 23, or to residues 19-443 of SEQ ID NO: 23, optionally further comprising an N-terminal sequence comprising residues 1-18 of SEQ ID NO: 23.   
     
     
         3 - 7 . (canceled) 
     
     
         8 . The recombinant glycoprotein or protein of  claim 1 , wherein the recombinant glycoprotein or protein: 
 (i) has an N-linked glycosylation pattern comprising core fucosylation;   (ii) has an overall length of no more than 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000 residues; and/or   (iii) lacks a functional endoplasmic reticulum retention signal, thereby enabling the formation of complex N-linked glycosylation in the Golgi apparatus of the  P. tricornutum  host cells.   
     
     
         9 - 11 . (canceled) 
     
     
         12 . An immunogenic composition (e.g., vaccine) comprising the recombinant glycoprotein or protein as defined in  claim 1 , and a suitable adjuvant. 
     
     
         13 . A  Phaeodactylum tricornutum  host cell that produces and/or preferably secretes the recombinant glycoprotein or protein as defined in  claim 1 , wherein the host cell comprises an exogenous expression cassette encoding the recombinant glycoprotein or protein operably linked to a promoter (e.g., an HASP1 promoter). 
     
     
         14 . A diagnostic device comprising the recombinant glycoprotein or protein as defined in  claim 1  for use in detecting the presence and/or concentration of antibodies that bind to said recombinant glycoprotein or protein. 
     
     
         15 . The diagnostic device of  claim 14 , which is a lateral flow test. 
     
     
         16 . (canceled) 
     
     
         17 . A method for triggering the production of antibodies against a coronavirus polypeptide antigen, the method comprising administering to a subject the immunogenic composition as defined in  claim 12 . 
     
     
         18 . A method for detecting antibodies specific to a coronavirus polypeptide antigen in a biological sample, the method comprising: (a) contacting the biological sample with the recombinant glycoprotein or protein as defined in  claim 1  ; and (b) detecting a complex formed between antibodies specific to the coronavirus polypeptide antigen and the recombinant glycoprotein or protein. 
     
     
         19 - 24 . (canceled) 
     
     
         25 . A method for producing a recombinant glycoprotein or protein, the method comprising:
 (a) providing  Phaeodactylum tricornutum  or other suitable host cells (e.g., diatom host cells or cells of the same clade of  P. tricornutum ) comprising a polynucleotide encoding the recombinant glycoprotein or protein in an expression cassette under control of an HASP1 (highly abundant secreted protein 1) promoter; and   (b) culturing the host cells in a production medium for a sufficient period of time to induce expression of the recombinant glycoprotein or protein, the production medium being maintained an inorganic phosphate concentration sufficiently low such that the recombinant glycoprotein or protein is expressed at a level higher than when the host cells are cultured under corresponding conditions in a phosphate-replete medium.   
     
     
         26 . The method of  claim 25 , wherein the production medium is a phosphate-reduced production medium having:
 (i) an inorganic phosphate concentration sufficiently low such that the recombinant glycoprotein or protein is expressed at a level at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2-fold higher than when the host cells are cultured under corresponding conditions in a phosphate-replete medium;   (ii) an inorganic phosphate concentration of less than or equal to 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of that present in a phosphate-replete growth medium that was used to culture the host cells provided in (a);   (iii) an inorganic phosphate concentration of less than or equal to: 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2.5, 2, 1.5, or 1 µM; or 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 ppm; or   (iv) any combination or (i) to (iii).   
     
     
         27 . The method of  claim 26 , wherein the host cells prior to (b) are cultured in a phosphate-replete growth medium having an inorganic phosphate concentration sufficiently high to repress expression of the recombinant glycoprotein or protein as compared to when the host cells are cultured in the phosphate-reduced production medium. 
     
     
         28 . The method of  claim 25 , wherein the production medium is a phosphate-reduced and iron-reduced production medium having an inorganic phosphate concentration as defined in  claim 25  and having:
 (i) an iron concentration sufficiently low such that the recombinant glycoprotein or protein is secreted at a level at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2-fold higher than when the host cells are cultured under corresponding conditions in an iron-replete medium; 
 (ii) an iron concentration of less than or equal to 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of that present in an iron-replete growth medium that was used to culture the host cells provided in (a); 
 (iii) an iron concentration of less than or equal to: 10, 9, 8, 7, 6, 5, 4, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.25, 0.2, 0.15, or 0.1 µM; 
 (iv) the recombinant protein is to be secreted from the host cells; or 
 (v) any combination or (i) to (iv). 
 
     
     
         29 . The method of  claim 26 , wherein the host cells prior to (b) are cultured in an iron-replete growth medium having an iron concentration sufficiently high to repress secretion of the recombinant glycoprotein or protein as compared to when the host cells are cultured in an iron-reduced production medium, or wherein the host cells prior to (b) are cultured in a phosphate-reduced and iron-reduced medium as defined in  claim 27  as a growth medium. 
     
     
         30 . The method of  claim 25 , wherein the host cells are cultured in the production medium for at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 days. 
     
     
         31 . The method of  claim 25 , wherein the host cells are engineered to comprise the expression cassette as part of their genome. 
     
     
         32 . The method of  claim 25 , wherein the recombinant glycoprotein or protein is heterologous with respect to the host cells and/or with respect to the HASP1 promoter. 
     
     
         33 - 39 . (canceled) 
     
     
         40 . A method for increasing secretion and/or expression of a recombinant glycoprotein or protein being expressed in an algae microorganism, said method comprising:
 a) providing a host cell algae microorganism comprising an expression cassette or vector comprising a polynucleotide encoding the recombinant glycoprotein or protein, and wherein the polynucleotide does not encode a Tobacco Etch Virus (TEV) protease cleavage site; and   b) culturing the host cells in a production medium for a sufficient period of time to induce expression and/or secretion of the recombinant glycoprotein or protein.   
     
     
         41 . The method of  claim 40 , wherein the algae microorganism is  Phaeodactylum tricornutum  or other suitable host cells (e.g., diatom host cells or cells of the same clade of  P. tricornutum ). 
     
     
         42 . The method of  claim 40 , wherein the polynucleotide further encodes a cleavable purification tag (e.g., glutathione-S-transferase (GST) tag, a histidine tag [e.g., 6His or 10His], or Fc tag). 
     
     
         43 - 45 . (canceled)